Advances in biotechnology have made possible the production of large amounts of therapeutically active and pure proteins and peptides. Currently, the therapeutic effects of most of these agents can be achieved only when they are administered via invasive routes, such as by injection. Since most proteins have very short half lives, effective concentrations of these agents can be maintained only when administered by frequent injections.
Although the administration of proteins by injection is the most effective means of their delivery in vivo, patient tolerance of multiple injections is very poor. In addition, drug injection requires training and skill that may not always be transferable to patients. In cases where protein drugs have a life-saving role, the administration by injection can be accepted by the patients. However, in cases where protein drugs are just one of several possible therapies, injections of proteins and peptides are unlikely to be accepted by the patients. Therefore, alternative routes of protein and peptide delivery need to be developed.
Such alternative routes may include the buccal, nasal, oral, pulmonary, rectal and ocular routes. Without exception, these routes are less effective than the parenteral routes of administration, but are still far more attractive than the parenteral routes because they offer convenience and control to the patients. The oral route is particularly attractive because it is the most convenient and patient-compliant.
Mucosal barriers, which separate the inside of the body from the outside (e.g. GI, ocular, pulmonary, rectal and nasal mucosa), comprise a layer of tightly joined cell monolayers which strictly regulate the transport of molecules. Individual cells in barriers are joined by tight junctions which regulate entry into the intercellular space. Hence, the mucosa is at the first level a physical barrier, transport through which depends on either the transcellular or the paracellular pathways [Lee, V. H. L., CRC. Critical Rev. Ther. Drug Delivery Sys., 5:69-97 (1988)].
Paracellular transport through water filled tight junctions is restricted to small molecules (MW &lt;1 kDa) and is essentially a diffusion process driven by a concentration gradient across the mucosa [Lee, (1988), supra; Artursson, P., and Magnusson, C., J. Pharm. Sci., 79:595-600 (1990)]. The tight junctions comprise less than 0.5% of the total surface area of the mucosa [Gonzalez-Mariscal, L. M. et al., J. Membrane. Biol., 86:113-125 (1985); Vetvicka, V., and Lubor, F., CRC Critical Rev. Ther. Drug Deliv. Sys., 5:141-170 (1988)]; therefore, they play only a minor role in the transport of protein drugs across the mucosa.
The transcellular transport of small drugs occurs efficiently provided the physiochemical properties of the drug are suited to transport across hydrophobic cell barriers. However, the transcellular transport of proteins and peptides is restricted to the process of transcytosis [Shen, W. C., et al., Adv. Drug Delivery Rev., 8:93-113 (1992)]. Transcytosis is a complex process in which proteins and peptides are taken up into vesicles from one side of a cell, and are subsequently shuttled through the cell to the other side of the cell, where they are discharged from the endocytic vesicles [Mostov, K. E., and Semister, N. E., Cell, 43:389-390 (1985)]. The cell membrane of mucosal barriers is a hydrophobic lipid bilayer which has no affinity for hydrophilic, charged macromolecules like proteins and peptides. In addition, mucosal cells may secrete mucin which can act as a barrier to the transport of many macromolecules [Edwards, P., British Med. Bull., 34:55-56 (1978)]. Therefore, unless specific transport mechanisms exist for protein and peptide, their inherent transport across mucosal barriers is almost negligible.
In addition to providing a tight physical barrier to the transport of proteins and peptides, mucosal barriers possess enzymes which can degrade proteins and peptides before, after, and during their passage across the mucosa. This barrier is referred to as the enzymatic barrier. The enzymatic barrier consists of endo- and exopeptidase enzymes which cleave proteins and peptides at their terminals or within their structure. Enzymatic activity of several mucosa have been studied and the results demonstrated that substantial protease activity exists in the homogenates of buccal, nasal, rectal and vaginal mucosa of albino rabbits and that these activities are comparable to those present in the ilium [Lee, et al., (1988), supra]. Therefore, regardless of the mucosa being considered, the enzymatic barrier present will feature strongly in the degradation of the protein and peptide molecules.
The N and the C termini of peptides are charged and the presence of charged side chains imparts highly hydrophilic characteristics on these macromolecules. In addition, the presence of charged side chains means that proteins and peptides have strong hydrogen bonding capacities; this H-bonding capacity has been demonstrated to play a major role in inhibiting the transport of even small peptides across cell membranes [Conradi, R. A., et al., Pharm. Res., 8:1453-1460 (1991)]. Therefore, the size and the hydrophilic nature of proteins and peptides combine to severely restrict their transport across mucosal barriers.
One approach that has been used to alter the physical nature of the mucosal barriers is the use of penetration enhancers. The use of penetration enhancers is based on the disruption of the cell barriers by low molecular weight agents which can fluidize cell membranes [Kaji, H., et al., Life Sci., 37:523-530 (1985)], open tight junctions [Inagaki, M., et al., Rhinology, 23:213-221 (1985)], and create pores in the cell membrane [Gordon, S., et al, Proc. Natl. Acad. Sci. USA, 82:7419-7423 (1985); Lee, V. H. L., et al, Crtical Reviews in Therapeutic Drug Camer Systems, CRC Press, 8:91-192 (1991)]. The use of these agents leads to a non-specific loss of barrier integrity and can lead to the absorption of a variety of large molecules which can be toxic to cells in vivo.
Protease inhibitors have been co-administered with proteins and peptides and have shown some limited activity in enhancing the absorption of these macromolecules in vivo [Kidron, M., et al., Life Sci., 31:2837-2841 (1982); Takaroi, K., et al., Biochem. Biophys. Res. Comm., 137:682-687 (1986)]. The safety and the long term effects of this approach have yet to be thoroughly investigated.
The prodrug approach is based on the modifications of peptides in a manner that will protect them from enzyme degradation and recognition. This has been achieved by substitution of the D-forms of amino acids in the structure of peptides, the blockage of vulnerable groups on peptides by amidation and acylation, the inversion of the chirality of peptides, and the introduction of conformational constraints in the peptide structure. The synthesis of prodrugs is only applicable to small peptides which have easily identifiable domains of activity.
Reduction in size is another feasible approach to increasing the transport potential of proteins. However, the active sites of proteins need to be mapped before size reduction can be attempted. In general, this approach is difficult to apply to the majority of proteins.
Carrier ligands, by virtue of their properties, can alter the cell uptake and transport characteristics of proteins and peptides. The essence of this approach is that a cell-impermeant protein or peptide is covalently attached to a carrier which is highly transported into cells. The mechanisms through which carrier ligands become endocytosed and transcytosed are important in deciding the suitability of the carrier for enhancing the transport of proteins and peptides. Macromolecular carriers are hydrophilic and do not partition into the membrane. Therefore, the transport of large polymeric carriers into the cells is mediated by the affinity of the carrier for the cell membrane. Generally, the uptake of a macromolecular conjugate starts with the binding to the cell membrane. The binding of the carrier to the cells can be specific (e.g. binding of antibodies to cell surface antigens), nonspecific (binding of cationic ligands or lectins to cell surface sugars), or receptor mediated (binding of transferrin or insulin to their receptors). Once the carrier is bound to the cell surface, it is taken up into vesicles. These vesicles then become processed stepwise and can be routed to several pathways. One pathway is the recycling of the vesicle back to the membrane from which it was invaginated. Another pathway, which is destructive to the conjugate, is the fusion with lysosomes. An alternative pathway, and one which leads to the transcytosis of the conjugate, is the fusion of the vesicle with the membrane opposite to the side from which it was derived.
The correct balance between the processes of endocytosis and transcytosis determine the delivery of a protein conjugate to its target. For instance, endocytosis may determine the extent to which a conjugate is taken up by the target cell, but transcytosis determines whether or not a conjugate reaches its target [Shen, et al., (1992), supra]. For successful absorption through the GI-tract, a conjugate must bind the apical membrane of the GI-mucosa, become internalized into the mucosal cells, be delivered across the cells, and finally become released from the basolateral membrane.
The current literature contains many reports which demonstrate that nonspecific carriers, such as polylysines [Shen, W. C. and Ryser, H. J. P., Proc. Natl. Acad. Sci. USA, 78:7589-7593 (1981)] and lectins [Broadwell, R. D., et al., Proc. Natl. Acad. Sci. USA, 85:632-646 (1988)], and specific carriers, such as transferrin [Wan, J., et al., J. Biol. Chem., 267:13446-13450 (1992)], asialoglycoprotein [Seth, R., et al., J. Infect Diseases, 168:994-999 (1993)], and antibodies [Vitetta, E. S., J. Clin. Immunol., 10:15S-18S (1990)] can enhance the endocytosis of proteins into cells. Reports dealing with transcytotic carriers for proteins are fewer, and very few studies have quantitated the transport of protein conjugates across cell barriers. Wheat germ agglutinin [Broadwell, et al., (1988), supra] and an anti-transferrin/methotrexate conjugate [Friden, P. M. and Walus, L. R., Adv. Exp. Med. Biol., 331:129-136 (1993)] have been shown to be transcytosed across the blood brain barrier in vivo. Also, polylysine conjugates of horseradish peroxidase (HRP) and a transferrin conjugate of HRP have been shown to be transcytosed across cell monolayers in vitro [Wan, J. and Shen, W. C., Pharm. Res., 8:S-5 (1991); Taub, M. E. and Shen, W. C., J. Cell. Physiol., 150:283-290 (1992); Wan, J., et al., J. Biol. Chem., 267:13446-13450 (1992), supra].
Fatty acids, as constituents of phospholipids, make up the bulk of cell membranes. They are available commercially and are relatively cheap. Due to their lipidic nature, fatty acids can easily partition into and interact with the cell membrane in a non-toxic way. Therefore, fatty acids represent potentially the most useful carrier ligands for the delivery of proteins and peptides. Strategies that may use fatty acids in the delivery of proteins and peptides include the covalent modification of proteins and peptides and the use of fatty acid emulsions.
Some studies have reported the successful use of fatty acid emulsions to deliver peptide and proteins in vivo [Yoshikawa, H., et al., Pharm. Res., 2:249-251 (1985); Fix, J. A., et al., Am. J. Physiol., 251:G332-G340 (1986)]. The mechanism through which fatty acid emulsions may promote the absorption of proteins and peptides is not yet known. Fatty acid emulsions may open tight junctions, solubilize membranes, disguise the proteins and peptides from the GI environment, and carry proteins and peptides across the GI-mucosa as part of their absorption [Smith, P., et al., Adv. Drug Delivery Rev., 8:253-290 (1992)]. The latter mechanism has been proposed, but is inconsistent with current knowledge about the mechanism of fat absorption.
A more logical strategy to deliver proteins and peptides across the GI-epithelium is to make use of fatty acids as non-specific membrane adsorbing agents. Several studies have shown that a non-specific membrane binding agent linked to a protein can promote the transcytosis of a protein conjugate across cells in vitro [Wan, J., et al., J. Cell. Physiol., 145:9-15 (1990); Taub and Shen (1992), supra]. Fatty acid conjugation has also been demonstrated to improve the uptake of macromolecules into and across cell membranes [Letsinger, R., et al., Proc. Natl. Aced. Sci. USA, 86:6553-4556 (1989); Kabanov, A., et al., Protein Eng., 3:39-42 (1989)]. Nonetheless, there have been difficulties in conjugating fatty acids to peptides and proteins, including: (1) the lack of solubility of fatty acids in the aqueous solution for the conjugation reaction; (2) the loss of biological activity of peptides and proteins after fatty acid acylation; and (3) the lack of solubilitty of fatty acid-conjugated peptides in aqueous solutions [see, e.g., Hashimoto, M., et al., Pharm. Res., 6:171-176 (1989); Martins, M. B. F., et al., Biochimie, 72:671-675 (1990); Muranishi, S., et al., Pharm. Res., 8:649-652 (1991); Robert, S., et al., Biochem. Biophys. Res. Commun., 196:447-454 (1993)].
It is an object of the present invention to provide methods and compositions for use in conjugating fatty acids to hydrophilic molecules and in improving the bioavailability of peptides and proteins.